The role of protein kinase C in MDRI gene transcription in multidrug resistant tumours

In this study the potential roles of PKC and on MDRI transcription regulation were explored. Human-derived MCF7 breast cancer cells that lack constitutive expression of PKC or at detectable levels, were transfected with full length PKC or genes driven by a Ponasterone A regulatable promoter. Stable transfectants were selected with appropriate antibiotics. Treatment of these cells with Ponasterone A induced PKC expression that was catalytically active and underwent translocation and downregulation on exposure to 12-O-tetradecanoyl-13-phorbol acetate (TPA). These cells were used to analyse PKC-mediated regulation of the MDRI promoter experimentally by transient transfection with 1073bp of the MDRI gene promoter, or deletion fragments to -8 bp each linked to a chloramphenicol acetyl transferase (CAT) reporter gene. In PKC expressing cells, TPA caused activation of all promoter fragments to -29 bp. These findings suggest that transcription may be mediated through the TPA-responsive EGR1 and Y-box motifs due to activation of PKC. Gel shift analysis demonstrated protein binding between -66 and -100 bp, indicating PKC-modified transcription factors regulate activity through the Y-box region. In contrast, PKC activated only two MDRI fragments, -982 and -612 bp. Gel shift studies showed that the transcription factor, AP1 might be responsible for regulating the -982 bp region. PKC and mediated activation was attenuated by the PKC inhibitor GF 109203X, suggesting that the MDRI transcription can be regulated by PKC and PKC. Microarray analysis demonstrated differential expression of genes that may be linked to MDRI transcriptional regulation via PKC-independent pathways. The results support the search for therapeutic strategies directed specifically against PKC, in order to overcome resistance of tumours against cytotoxic agents.